Introduction

The goal of this project was to perform RNA-seq based differential gene expression analysis of Zebrafish embryos to characterize the response to potential neuroprotective agents in a chemical model of neurodegeneration, beta-methylamino-L-alanine (BMAA) toxin.

Experiment Design

3 replicates for each of the 5 conditions below were prepared and each RNA-seq library was sequenced at an average depth of 24.3 million read pairs: - Control - untreated zebrafish embryos - BMAA - zebrafish embryos given BMAA toxin to induce neurodegenerative symptoms - CNR-401 - toxified zebrafish embryos subsequently given CNR-401 as treatment - Pure Cannflavin A (CNR-402) - toxified zebrafish embryos subsequently given pure cannflavin A as treatment - Edaravone - toxified zebrafish embryos subsequently given Edaravone as treatment

Raw reads were trimmed and mapped to the zebrafish reference genome with an average of 19.8 million read pairs mapped per sample

Pair-wise differential gene expression analysis was done using DESeq2 for all 10 sample pairs, producing differential gene expression (DGE) data.

The structure of the files is shown below using the BMAA vs BMAA-CNR-401 file as an example.

Each file starts with the same number of genes: 28788

Adding a column, “Expression”, to signify whether a gene was upregulated or downregulated for additional context in the volcano plots. Significance is defined by an absolute log2 fold change greater than or equal to 0.5 AND an adjusted p-value less than 0.05.

Data Analysis

The volcano plots below display the results of RNA-seq experiments to show the statistical significance (adjusted P value) versus magnitude of change (log 2 fold change). The CNR-401 plot shows a vast number of differentially expressed genes (DEG) following treatment. Genes that are not significant are shown as grey points.

Using the DGE comparisons of BMAA vs BMAA-401, BMAA vs BMAA-Edaravone, and BMAA vs BMAA-Pure, differential expression was screened for biological and statistical significance using a adjusted p-value cutoff of 0.05, and a log2 fold change cutoff of 0.5.

The number of genes remaining in each file are:
- BMAA vs BMAA-CNR-401: 4576
- BMAA vs BMAA-Edaravone: 893
- BMAA vs BMAA-PureCFA: 528
- BMAA vs Control: 2001

GProfiler was used to map significant DEGs in each DGE comparison file to human orthologs.

BMAA vs BMAA-CNR-401

BMAA vs BMAA-Edaravone

BMAA vs BMAA-PureCFA

BMAA vs Control

The Venn diagrams below represent the number of significantly differentially expressed genes after BMAA induction and after treatment (BMAA + CNR-401 / BMAA + Edaravone). A total of 3073 genes differentially expressed with CNR-401 have human orthologs; 670 genes differentially expressed with Edaravone have human orthologs

Gene set enrichment analysis of the human orthologs of significant genes produces the following results:

BMAA vs BMAA-CNR-401

BMAA vs BMAA-Edaravone

BMAA vs Control

The following bar plots show the most significant biological process GO terms, i.e., with the lowest adjusted p-values.

The following bar plots include select GO terms shared by CNR-401 and Edaravone.

The bar plot below includes significant GO terms unique to CNR-401.